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An ouabain-like factor has been implicated repeatedly in salt-sensitive hypertension as a natriuretic agent. However, the response of plasma ouabain-like factor to acute and chronic variation of body sodium is unclear. We studied 138 patients with essential hypertension who underwent an acute volume expansion/contraction maneuver (2 days) and 20 patients who entered a blind randomized crossover design involving chronically controlled sodium intake and depletion (170 to 70 mmol/d; 2 weeks each period). In both studies, plasma levels of ouabain-like factor were higher during sodium depletion (acute: 338.8±17.4 and 402.7±22.8 pmol/L for baseline and low sodium, respectively, P P =0.01). No significant change in plasma ouabain-like factor was observed after a 2-hour saline infusion (333.4±23.9 pmol/L) or controlled sodium (402.1±34.9 pmol/L). When patients were divided into salt-sensitive or salt-resistant groups, no differences in plasma ouabain-like factor were observed in the 2 groups at baseline or in response to the 2 protocols: salt resistant (n=69, 340.1±25.9 pmol/L) versus salt sensitive (n=69, 337.4±23.6 pmol/L) and chronic salt resistant (n=11, 336.0±53.2) versus salt sensitive (n=9, 301.1±331.4 pmol/L). However, circulating ouabain-like factor was increased by sodium depletion in both groups. These results demonstrate that circulating ouabain-like factor is raised specifically by maneuvers that promote the loss of body sodium. Acute expansion of body fluids with isotonic saline is not a stimulus to plasma ouabain-like factor. Moreover, basal levels of plasma ouabain-like factor do not differ among patients with salt-sensitive or salt-resistant hypertension. Taken together, these new results suggest that ouabain-like factor is involved in the adaptation of humans to sodium depletion and argue against the hypothesis that ouabain-like factor is a natriuretic hormone.

Aims Diabetic cardiomyopathy is a multifactorial disease characterized by an early onset of diastolic dysfunction (DD) that precedes the development of systolic impairment. Mechanisms that can restore cardiac relaxation improving intracellular Ca2+ dynamics represent a promising therapeutic approach for cardiovascular diseases associated to DD. Istaroxime has the dual properties to accelerate Ca2+ uptake into sarcoplasmic reticulum (SR) through the SR Ca2+ pump (SERCA2a) stimulation and to inhibit Na+/K+ ATPase (NKA). This project aims to characterize istaroxime effects at a concentration (100 nmol/L) marginally affecting NKA, in order to highlight its effects dependent on the stimulation of SERCA2a in an animal model of mild diabetes. Methods and results Streptozotocin (STZ) treated diabetic rats were studied at 9 weeks after STZ injection in comparison to controls (CTR). Istaroxime effects were evaluated in vivo and in left ventricular (LV) preparations. STZ animals showed 1) marked DD not associated to cardiac fibrosis, 2) LV mass reduction associated to reduced LV cell dimension and T-tubules loss, 3) reduced LV SERCA2 protein level and activity and 4) slower SR Ca2+ uptake rate, 5) LV action potential (AP) prolongation and increased short-term variability (STV) of AP duration, 6) increased diastolic Ca2+, and 7) unaltered SR Ca2+ content and stability in intact cells. Acute istaroxime infusion (0.11 mg/kg/min for 15 min) reduced DD in STZ rats. Accordingly, in STZ myocytes istaroxime (100 nmol/L) stimulated SERCA2a activity and blunted STZ-induced abnormalities in LV Ca2+ dynamics. In CTR myocytes, istaroxime increased diastolic Ca2+ level due to NKA blockade albeit minimal, while its effects on SERCA2a were almost absent. Conclusions SERCA2a stimulation by istaroxime improved STZ-induced DD and intracellular Ca2+ handling anomalies. Thus, SERCA2a stimulation can be considered a promising therapeutic approach for DD treatment. Translational perspective Deficient sarcoplasmic reticulum (SR) Ca2+ uptake has been identified in cardiomyocytes from failing human hearts with impaired diastolic relaxation (e.g. diabetic hearts) and has been associated with a decreased SERCA2a expression and activity and/or with a higher SERCA2a inhibition by phospholamban. Thus, SERCA2a may represent a pharmacological target for interventions aimed at improving cytosolic Ca2+ compartmentalization into the SR to limit diastolic dysfunction pathologies. In this context, istaroxime is the first-in-class luso-inotropic agent targeting SERCA2a that has already demonstrated its efficacy in clinical trials and may be useful to clarify the relevance of SERCA2a stimulation in controlling cytosolic Ca2+ level.

Glomerulopathies are important causes of morbidity and mortality. Selective therapies that address the underlying mechanisms are still lacking. Recently, two mechanisms, mutant β-adducin and ouabain, have been found to be involved in glomerular podocytopathies and proteinuria through nephrin downregulation. The main purpose of the present study was to investigate whether rostafuroxin, a novel antihypertensive agent developed as a selective inhibitor of Src-SH2 interaction with mutant adducin- and ouabain-activated Na,K-ATPase, may protect podocytes from adducin- and ouabain-induced effects, thus representing a novel pharmacologic approach for the therapy of podocytopathies and proteinuria caused by the aforementioned mechanisms. To study the effect of rostafuroxin on podocyte protein changes and proteinuria, mice carrying mutant β-adducin and ouabain hypertensive rats were orally treated with 100 μg/kg per day rostafuroxin. Primary podocytes from congenic rats carrying mutant α-adducin or β-adducin (NB) from Milan hypertensive rats and normal rat podocytes incubated with 10(-9) M ouabain were cultured with 10(-9) M rostafuroxin. The results indicated that mutant β-adducin and ouabain caused podocyte nephrin loss and proteinuria in animal models. These alterations were reproduced in primary podocytes from NB rats and normal rats incubated with ouabain. Treatment of animals, or incubation of cultured podocytes with rostafuroxin, reverted mutant β-adducin- and ouabain-induced effects on nephrin protein expression and proteinuria. We conclude that rostafuroxin prevented podocyte lesions and proteinuria due to mutant β-adducin and ouabain in animal models. This suggests a potential therapeutic effect of rostafuroxin in patients with glomerular disease progression associated with these two mechanisms.

Mutant α-adducin and endogenous ouabain levels exert a causal role in hypertension by affecting renal Na-K ATPase. In addition, mutant β-adducin is involved in glomerular damage through nephrin down-regulation. Recently, the salt-inducible kinase 1 (SIK1) has been shown to exert a permissive role on mutant α-adducin effects on renal Na-K ATPase activity involved in blood pressure (BP) regulation and a SIK1 rs3746951 polymorphism has been associated with changes in vascular Na-K ATPase activity and BP. Here, we addressed the role of SIK1 on nephrin and glomerular functional modifications induced by mutant β-adducin and ouabain, by using congenic substrains of the Milan rats expressing either mutant α- or β-adducin, alone or in combination, ouabain hypertensive rats (OHR) and hypertensive patients. SIK1 co-localized and co-immunoprecipitated with nephrin from glomerular podocytes and associated with caveolar nephrin signaling. In cultured podocytes, nephrin-gene silencing decreased SIK1 expression. In mutant β-adducin congenic rats and in OHR, the podocyte damage was associated with decreased nephrin and SIK1 expression. Conversely, when the effects of β-adducin on podocytes were blocked by the presence of mutant α-adducin, nephrin and SIK1 expressions were restored. Ouabain effects were also reproduced in cultured podocytes. In hypertensive patients, nephrinuria, but not albuminuria, was higher in carriers of mutant SIK1 rs3746951 than in wild-type, implying a more direct effect of SIK1 on glomerular damage. These results demonstrate that, through nephrin, SIK1 is involved in the glomerular effects of mutant adducin and ouabain and a direct effect of SIK1 is also likely to occur in humans.

Recent evidence of beneficial effects of ranolazine (RAN) in type II diabetes motivates interest in the role of the late sodium current (INaL) in glucose-stimulated insulin secretion. In the present work, we characterize INaL and its function in rat INS-1E cells and human islets cells. INaL was identified as steady-state current blocked by 10 μM RAN (IRAN) or 0.5 μM tetrodotoxin (TTX) (ITTX). Veratridine (VERA, 40 μM) was used as INaL enhancer. Baseline INaL was similar between INS-1E and human islet cells. In INS-1E cells, activated by glucose or tolbutamide, TTX or RAN hyperpolarized membrane potential (V m). VERA-induced depolarization was countered by TTX or RAN. ITTX and IRAN reversal potentials were negative to Na+ equilibrium one, but they approached it after Na+ substitution with Li+ or when K+ channels were blocked. This revealed INaL coupling with Na+-activated K+ current (IKNa); expression of IKNa channels (Slick/Slack) was confirmed by transcript analysis and Western blot. RAN or TTX blunted cytosolic Ca2+ response to depolarization. Long-term incubation in high (33 mM) glucose (CHG) constitutively enhanced INaL. VERA immediately increased glucose-stimulated insulin secretion. CHG increased glucose-independent secretion instead and abolished the secretory response to glucose. RAN or TTX countered VERA- and CHG-induced changes in insulin secretion. Our study demonstrated that (1) INaL was expressed in insulin-secreting cells and coupled to IKNa; INaL affected cytosolic Ca2+ but, unless enhanced, barely contributed to glucose-stimulated insulin secretion (GSIS); and (2) sustained hyperglycemic stress enhanced INaL, which contributed to the attending increase of glucose-independent insulin “leak” and GSIS impairment.

Endogenous ouabain-like factor (OLF) is present in mammal tissues and after standardized extraction procedure can be similarly quantified by two independent assays: RIA and Na-KATPase inhibition. OLF was quantified both from plasma and tissues obtained from MHS hypertensive and MNS normotensive rats, maintained under the same environmental and dietary conditions, and from plasma of healthy volunteers and essential hypertensive patients. OLF biochemical characterization shows that it behaves like ouabain except for a 1000-fold higher affinity for the ouabain low-affinity Na-KATPase isoforms than ouabain. Tissue and plasma levels of OLF are higher in MHS than in MNS rats and are not influenced by exogenous OLF sources. Plasma OLF is also increased in a subgroup of hypertensive patients. Both in rats and humans a primary cell membrane alteration affecting ion transports seems to be linked to the increased levels of OLF. An antihypertensive compound which selectively antagonizes the pressor effect of OLF and corrects the ion transport defect is under development and can represent a new pharmacological approach to the treatment of hypertension.

Hypertension is a prevalent disorder in the world representing one of the major risk factors for heart attack and stroke. These risks are increased in salt sensitive individuals. Hypertension and salt sensitivity are complex phenotypes whose pathophysiology remains poorly understood and, remarkably, salt sensitivity is still laborious to diagnose. Here we present a urinary proteomic study specifically designed to identify urinary proteins relevant for the pathogenesis of hypertension and salt sensitivity. Despite previous studies that underlined the association of UMOD gene variants with hypertension, this work provides novel evidence showing different uromodulin protein level in the urine of hypertensive patients compared to healthy individuals. Notably, we also show that patients with higher level of uromodulin are homozygous for UMOD risk variant and display a decreased level of salt excretion, highlighting the essential role of UMOD in the regulation of salt reabsorption in hypertension. Additionally, we found that urinary nephrin 1, a marker of glomerular slit diaphragm, may predict a salt sensitive phenotype and positively correlate with increased albuminuria associated with this type of hypertension., Highlights • We identified urinary proteins differently excreted in hypertensive patients. • Nephrin 1 might predict salt sensitive phenotype and glomerular complications. • Uromodulin impacts salt homeostasis in hypertension. • We provide new insights into the pathogenesis of hypertension and salt sensitivity.

Abstract The resolution of controversies that concern the detectability of an endogenous ouabain-like factor (OLF) in mammalian tissues and plasma was approached by the application of a standardized method for its extraction and quantification. Two independent assays were used to quantify the OLF: (1) a radioimmunoassay, which used a polyclonal antiouabain antiserum, and (2) a radioenzymatic assay based on the inhibition of dog kidney Na + ,K + -ATPase. Plasma and tissues were obtained from the Milan hypertensive strain (MHS) and the Milan normotensive strain (MNS) of rats and from healthy human volunteers. Results indicate that (1) a single high-performance liquid chromatography (HPLC) fraction identical to that of ouabain was identified by both assay methods in the rat hypothalamus and hypophysis and in both rat and human plasma; (2) dilution curves of OLF and standard ouabain were parallel and with a similar K d , both in radioimmunoassay (3 nmol/L) and ATPase assay (14 nmol/L); (3) after HPLC, OLF was similarly quantified by the two methods in the hypothalamus, hypophysis, adrenals, and plasma of rats and in human plasma; (4) OLF was present in larger amounts in the hypothalamus, hypophysis, and plasma of MHS rats than that of MNS rats; (5) the HPLC fraction of human plasma was quantified similarly by both assays (range, 60 to 150 pmol/L); (6) recovery of standard ouabain in pre-HPLC plasma extracts was approximately 90%; and (7) pre-HPLC OLF concentrations in human plasma ranged between 0.05 and 0.75 nmol/L. Rat cerebral tissues and both rat and human plasma contained measurable amounts of OLF, which were quantified similarly by radioimmunoassay and ATPase assay, both before and after HPLC fractionation. The increased MHS tissue and plasma levels of OLF are in keeping with the pathogenetic role of this factor in MHS hypertension.

Milan hypertensive rats (MHS) develop hypertension because of a primary renal alteration. Both apical and basolateral sodium transport are faster in membrane vesicles derived from renal tubules of MHS than in those of Milan normotensive control rats (MNS). These findings suggest that the increased renal sodium retention and concomitant development of hypertension in MHS may be linked to an altered transepithelial sodium transport. Since this transport is mainly under the control of the Na-K pump, we investigated whether an alteration of the enzymatic activity and/or protein expression of the renal Na,K-ATPase is detectable in prehypertensive MHS. We measured the Na,K-ATPase activity, Rb + occlusion, turnover number, α 1 - and β 1 -subunit protein abundance, and α 1 and β 1 mRNA levels in microsomes from renal outer medulla of young (prehypertensive) and adult (hypertensive) MHS and in age-matched MNS. In both young and adult MHS, the Na,K-ATPase activity was significantly higher because of an enhanced number of active pump sites, as determined by Rb + occlusion maximal binding. The higher number of pump sites was associated with a significant pretranslational increase of α 1 and β 1 mRNA levels that preceded the development of hypertension in MHS. Since a molecular alteration of the cytoskeletal protein adducin is genetically associated with hypertension in MHS and is able to affect the actin-cytoskeleton and Na-K pump activity in transfected renal cells, we propose that the in vivo upregulation of Na-K pump in MHS is primary and linked to a genetic alteration of adducin.

Twenty years of genetic studies have not contributed to improvement in the clinical management of primary arterial hypertension. Genetic heterogeneity, epistatic-environmental-biological interactions, and the pathophysiological complexity of hypertension have hampered the clinical application of genetic findings. In the companion article, we furnished data from rodents and human cells demonstrating two hypertension-triggering mechanisms-variants of adducin and elevated concentrations of endogenous ouabain (within a particular range)-and their selective inhibition by the drug rostafuroxin. Here, we have investigated the relationship between variants of genes encoding enzymes for ouabain synthesis [LSS (lanosterol synthase) and HSD3B1 (hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 1)], ouabain transport {MDR1/ABCB1 [ATP-binding cassette, sub-family B (MDR/TAP), member 1]}, and adducin activity [ADD1 (adducin 1) and ADD3], and the responses to antihypertensive medications. We determined the presence of these variants in newly recruited, never-treated patients. The genetic profile defined by these variants predicted the antihypertensive effect of rostafuroxin (a mean placebo-corrected systolic blood pressure fall of 14 millimeters of mercury) but not that of losartan or hydrochlorothiazide. The magnitude of the rostafuroxin antihypertensive effect was twice that of antihypertensive drugs recently tested in phase 2 clinical trials. One-quarter of patients with primary hypertension display these variants of adducin or concentrations of endogenous ouabain and would be expected to respond to therapy with rostafuroxin. Because the mechanisms that are inhibited by rostafuroxin also underlie hypertension-related organ damage, this drug may also reduce the cardiovascular risk in these patients beyond that expected by the reduction in systolic blood pressure alone. ispartof: Science Translational Medicine vol:2 issue:59 ispartof: location:United States status: published

A series of compounds related to torasemide, a loop diuretic, were synthesized and examined for their diuretic potency and inhibitory activity on the erythrocyte and renal medullary thick ascending limb vesicle Na+,2Cl-,K+ cotransport in Milan hypertensive (MHS) and normotensive (MNS) rat strains, where previous studies had demonstrated an alteration of the cotransport system genetically related to hypertension. From the results of the screening, structure-activity relationships were drawn and two compounds, JDL 961 and C 2921 were selected. Their IC50 on renal vesicle cotransport were similar in the two strains (JDL 961: MHS = 1.8 microM; MNS = 1.2 microM; C 2921: MHS = 4 microM; MNS = 3.8 microM), and were 4-8 times lower than those of torasemide (MHS = 13 microM; MNS = 31 microM, P less than 0.01) and 50-60 times lower than those of bumetanide (MHS = 145 microM; MNS = 206 microM, P less than 0.05) taken as reference compounds. Their ability to reduce the development rate of hypertension was tested both in MHS and in Okamoto spontaneously hypertensive rats (SHR) strain, in which cotransport alterations are opposite to those of MHS. Both torasemide derivatives (7.5 mg.kg-1 os per day) prevented development of hypertension in the two strains. The time course of this hypotensive activity was faster and the percentage of blood pressure fall greater in MHS (20-25%) than in SHR rats (12-15%), even though the absolute value of blood pressure fall was similar in MHS (JDL 961 = -17 mm Hg; C 2921 = -30 mm Hg) and SHR (JDL 961 = -25 mm Hg; C 2921 = -20 mm Hg). A superimposable effect of bumetanide was observed in the two strains, but at 8 times higher daily dose (60 mg.kg-1). These results suggest that new loop diuretics can be selected for their antihypertensive activity on the basis of their in vitro potency in inhibiting the Na+,2Cl-,K+.

An endogenous ouabain has been isolated and conclusively identified from several mammalian tissues, including human plasma, by a number of independent laboratories. Substantial evidence from independent laboratories in several continents is consistent with an adrenal source for most if not all of the circulating endogenous ouabain. Accumulating evidence suggests that circulating levels of endogenous ouabain in humans are modulated by dietary salt and chronic volume status. Endogenous ouabain is linked significantly with vascular function in hypertension and likely impacts the pathogenesis of heart and renal failure. Moreover, the molecular mechanism of endogenous ouabain-linked hypertension involves the sodium pump/sodium-calcium exchange duet. The outstanding analytical issues include the elucidation of distal events in the biosynthetic pathway for endogenous ouabain and identification of molecular mechanisms that regulate its secretion and clearance.

This paper describes experiments to examine Rb + fluxes via the Na + /K + /Cl − cotransporter in membrane vesicles from renal outer medulla of three strains of rat: (A) Wistar (B) Milan hypertensive (MHS) and normotensive (MNS), and (C) Sabra salt-sensitive hypertensive (SBH) and salt-resistant (SBN). Initially, Na + -dependent furosemide- or bumetanide-inhibited 86 Rb + fluxes were characterised using Wistar rat microsomes. The latter were partially purified on a metrizamide cushion, and assay conditions were optimized for use with microsomes from the other rats. The major result is that in microsomes from adult Milan hypetensive (MHS) rats the rate of the Na + /K + /Cl − -cotransporter mediated 86 Rb flux at sub-saturating concentrations of Rb, appears to be significantly greater than in the normotensive (MNS) controls. The effect reflects an increased apparent Rb affinity of the cotransporter in MHS microsomes. There is no difference in maximal rate or in the apparent Na + activation affinity of the 86 Rb + flux. In addition bumetanide appears to be a somewhat more effective inhibitor in MHS compared to MNS microsomes. The 86 Rb + flux result is compatible with a previous finding that in red cells, Na + /K + -cotransporter mediated fluxes are increased in MHS compared to MNS. It supports the notion that the Na + /K + /Cl − -cotransporter in both red cells and kidney is a genetic marker for hypertension. It is of interest that apparetly more than one Na + transport system is affected in MHS hypertensive kidneys (a) the Na + /K + /Cl − cotransporter i nthe thick ascending limb of Henle and (b) the Na + /H + exchanger and/or a conductive Na + -pathway in brush-border membranes from proximal tubule. It is conceivable that in the hypertensive animals a common regulatory pathway (e.g., phosphorylation) or protein (e.g., cytoskeleton) is affected along the length of the nephron. In Sabra SBH and SBN rat microsomes, no difference was found for the 86 Rb + flux via the Na + /K + /Cl − cotransporter (or via a K + channel).

Hypertension is an important public health problem affecting more than 50 million individuals in the USA alone. The most common form, essential hypertension, results from the complex interplay between genetic predisposition and environmental influences. Epidemiological, migration, intervention and genetic studies in humans and animals provide very strong evidence of a causal link between high salt intake and high blood pressure. One of the candidate genes for salt-sensitive hypertension is adducin. Adducin is a heterodimeric cytoskeleton protein, the three subunits of which are encoded by genes (ADD1, ADD2 and ADD3) that map to three different chromosomes. A long series of parallel studies in the Milan hypertensive rat strain model of hypertension and humans indicated that an altered adducin function might cause hypertension through enhanced constitutive tubular sodium reabsorption. An example of a prospective efficacy of pharmacogenetics and pharmacogenomics is the detection and impact of adducin polymorphisms on hypertension. In particular, the selective advantages of diuretics in preventing myocardial infarction and stroke over other antihypertensive therapies that produce a similar blood pressure reduction in carriers of the mutated adducin may support new strategies aimed at optimizing the use of new antihypertensive agents for the prevention of hypertension-associated organ damage.

Essential hypertension is a heterogeneous multifactorial syndrome associated with a high cardiovascular risk. A multiple choice of antihypertensive drugs is available; however, a high individual variability to the antihypertensive therapy is still responsible for a modest reduction of the CV risk and not satisfactory control of blood pressure levels. The success of future hypertension treatment will depend upon the understanding of the genetic molecular mechanisms operating in subsets of patients, and the ability of new drugs to specifically correct such alterations. Two mechanisms, among others, are involved in determining the abnormalities of tubular Na(+) reabsorption observed in essential hypertension: the polymorphism of the cytoskeletal protein alpha-adducin and the increased circulating levels of endogenous ouabain (EO). Both lead to increased activity and expression of the renal Na-K pump, the driving force for tubular Na transport. Morphological and functional cardiovascular alterations have also been associated with adducin and EO. Rostafuroxin is a new oral antihypertensive agent able to selectively antagonize adducin and EO hypertensive and molecular effects. It is endowed with high potency and efficacy in reducing blood pressure and preventing organ hypertrophy in animal models representative of both adducin and EO mechanisms. At molecular level, in the kidney, Rostafuroxin normalizes the enhanced activity of the Na-K pump induced by adducin mutation and antagonizes the EO triggering of the Src-EGFr-dependent signaling pathway leading to renal Na-K pump, and ERK Tyrosin phosphorylation and activation. In the vasculature, it normalizes the increased myogenic tone caused by ouabain. A very high safety ratio and an absence of interaction with other mechanisms involved in blood pressure regulation, together with initial evidence of high tolerability and efficacy in hypertensive patients, indicate Rostafuroxin as the first example of a new class of antihypertensive agents designed to antagonize adducin and EO-hypertensive mechanisms. Currently, a phase II multicenter European clinical trial is ongoing for providing the proof of concept that such a compound is effective in the subset of patients where these two mechanisms are at work.

Elevated levels of the endogenous ouabain (EO), a closely related isomer of ouabain, are implicated in rat and human hypertension and in related cardiovascular complications. The pathogenetic mechanisms through which EO affects the cardiovascular system involve the modulation of the renal Na/K-ATPase, implicated in renal tubular sodium reabsorption, and the activation of signal transduction pathways, promoting the transcription of growth-related genes. Experimental and clinical evidence on rats and humans stimulated the pharmacological research for developing novel anti-hypertensive agents able to antagonize the cellular and molecular alterations mediated by EO. Among them, the digitoxigenin derivate, PST 2238, has been selected for its ability to antagonize the ouabain-induced effects on blood pressure and organ hypertrophy at oral doses of microgram/kg/day in vivo. The pharmacological selectivity and safety of PST 2238 suggests that the compound may be effective for the treatment of those forms of hypertension in which renal sodium handling alterations and cardiovascular complications are associated with increased production of EO.

Abstract —Many patients with essential hypertension (EH) exhibit increased left ventricular mass. Similarly, elevated circulating levels of an endogenous ouabainlike factor (OLF) have been described in some but not all patients with EH. Moreover, ouabain has a hypertrophic influence on isolated cardiac myocytes. Accordingly, we investigated relationships among plasma OLF, left ventricular mass, and cardiac function in patients with EH. Plasma OLF was determined in 110 normotensive subjects and 128 patients with EH. Echocardiographic parameters and humoral determinants were measured in EH. Plasma OLF levels were increased ( P P =0.03). Left ventricular mass index and stroke volume in EH with high OLF were greater than in EH with normal OLF (101.9±3.3 versus 86.1±2.5 g/m 2 , P =0.0003, and 57.10±1.48 versus 52.30±1.14 mL/m 2 , P =0.02, respectively), although heart rate was slower (74.2±1.3 versus 80.5±1.3 bpm, P =0.005). Multiple regression analysis that tested the influence of body mass index, age, gender, 24-hour blood pressure, and OLF on left ventricular mass revealed independent contributions of systolic (13.2%) and diastolic (12.4%) blood pressure and plasma OLF (11.6%) to left ventricular mass. We conclude that ≈50% of patients with uncomplicated EH have elevated-high circulating OLF levels, higher diastolic blood pressure, greater left ventricular mass and stroke volume, and reduced heart rate. We propose that the OLF affects cardiovascular function and structure and should be considered as a factor that contributes to the risk of morbid events.

The effect of ethylisopropyl-amiloride (EIPA) and phenamil on sodium uptake in renal brush border membrane vesicles from prehypertensive rats of the Milan strain (MHS) and their normotensive controls (MNS) was investigated. In the presence of both a membrane potential and a pH gradient a differential effect of EIPA and phenamil was evidenced between the two rat strains. In the absence of a pH gradient, but in the presence of a membrane potential, EIPA was about two-fold more potent than phenamil in inhibiting sodium transport in both rat strains, excluding the presence of epithelial sodium channels in our BBMV preparations. Taken together these results support the hypothesis that a structurally different Na + H + exchanger located on the brush border membrane may be involved in the increased tubular sodium reabsorption observed in vivo in hypertensive rats.

The (Na+,K+)-ATPase activity from the kidney cortex of the Milan hypertensive rat strain (MHS) and the corresponding normotensive control (MNS) was measured both in active solubilized enzyme preparations and in isolated basolateral membrane vesicles. Kinetic analysis of the purified enzyme showed that the Vmax value was significantly higher in MHS rats. The difference between MHS and MNS was not linked to a different number of sodium pumps, but was related to the molecular activity of the enzyme. Using basolateral membrane vesicles, an increased ATP-dependent ouabain-sensitive sodium transport was also demonstrated in MHS rats. These results support the hypothesis that a higher tubular sodium reabsorption may be involved in the pathogenesis of hypertension in this rat strain.

An approach to evaluating the genetic components of essential hypertension using an animal model, the Milan hypertensive strain (MHS) of rats, and studies in human families with positive and negative histories for high blood pressure are described and discussed. Differences at renal and cellular levels between MHS and its normotensive control strain, MNS, show many similarities to those between offspring from hyper- and normotensive families in humans. These include, with respect to the former group of each species, lower erythrocyte volume and Na content, higher Na-K cotransport across red blood cell (RBC) membranes higher Na excretion after load, and greater pressor effect in transplanted kidneys. A novel protein found in rat RBC cytoskeleton appears to have a function in Na-K cotransport and it may, eventually, be possible to demonstrate in man.

Defective pressure-natriuresis related to abnormalities in the natriuretic response has been associated with hypertension development. A major signaling pathway mediating pressure natriuresis involves the cGMP-dependent protein kinase 1 (PRKG1) that, once activated by Src kinase, inhibits renal Na + reabsorption via a direct action on basolateral Na-K ATPase and luminal Na–H exchanger type 3, as shown in renal tubuli of animals. Because a clear implication of PRKG1 in humans is still lacking, here we addressed whether PRKG1 polymorphisms affect pressure-natriuresis in patients. Naive hypertensive patients (n=574), genotyped for PRKG1 rs1904694, rs7897633, and rs7905063 single nucleotide polymorphisms (SNPs), underwent an acute Na + loading, and the slope of the pressure–natriuresis relationship between blood pressure and Na + excretion was calculated. The underlying molecular mechanism was investigated by immunoblotting protein quantifications in human kidneys. The results demonstrate that the PRKG1 risk haplotype GAT (rs1904694, rs7897633, rs7905063, respectively) associates with a rightward shift of the pressure–natriuresis curve (0.017±0.004 μEq/mm Hg per minute) compared with the ACC (0.0013±0.003 μEq/mm Hg per minute; P =0.001). In human kidneys, a positive correlation of protein expression levels between PRKG1 and Src ( r =0.83; P r =0.557; P r =0.584; P r =0.691; P + reabsorption, suggestive of a blunt pressure–natriuresis response.